Chapter 8 – Timing of Embryo Culture


Following the fusion of the oocyte and spermatozoon and successful oocyte activation, preimplantation development begins with the formation of the zygote, displaying two pronuclei and two polar bodies if correctly fertilized (Figure 8.1). Embryo development continues with the onset of cleavage, i.e., consecutive mitotic divisions, leading to a major wave of embryonic genome activation between the 4- and 8-cell stages on Day 3 of development.

Chapter 8 Timing of Embryo Culture

Ioannis Sfontouris

8.1 Preimplantation Embryo Development

Following the fusion of the oocyte and spermatozoon and successful oocyte activation, preimplantation development begins with the formation of the zygote, displaying two pronuclei and two polar bodies if correctly fertilized (Figure 8.1). Embryo development continues with the onset of cleavage, i.e., consecutive mitotic divisions, leading to a major wave of embryonic genome activation between the 4- and 8-cell stages on Day 3 of development.

Figure 8.1 Preimplantation embryo development. (Photographs taken by the author).

On Day 4, the embryo undergoes compaction, followed by cavitation and the formation of a fluid-filled cavity, marking the formation of an early blastocyst. The blastocyst continues to expand through mitotic divisions and the enlargement of the blastocoel, which occupies almost the entire embryo volume at the expanded stage by Day 5. The combined effect of increasing pressure from the expanding blastocyst on the zona pellucida and the action of embryo-secreted enzymes leads to a breach of the zona pellucida and the progressive escape of the embryo proper, until it hatches fully in preparation for implantation in the uterine endometrium.1

Development rate is considered the most important indicator of embryo viability.2 For example, on Day 3 (68 ± 1 hour post insemination), embryos with ≤6 cells are considered slow, embryos with 7–9 cells normal, and embryos with ≥10 cells are considered accelerated.3 There is evidence to suggest that too slow or too fast embryo cleavage has a negative impact on implantation rate. In addition, embryos with deviations in cleavage rate are associated with a higher incidence of chromosomal abnormalities compared with normally developing embryos.4

Standardized timing of evaluations performed at set time points (hours post insemination/microinjection) enables development rates and results to be compared between different embryo cohorts and different laboratories.3 (See Table 8.1). However, it must be noted that these recommendations were made based on static observations of embryos once or twice daily. The introduction of time-lapse technology has provided a wealth of new information. A better knowledge of timings and intervals between cell divisions, as well as the possibility of identifying abnormal cleavage patterns, such as direct or reverse cleavage, are likely to lead to a revision of the criteria for embryo assessment.

Table 8.1 Recommendations of the Istanbul Consensus regarding the timing of observation of fertilized oocytes and embryos, and the expected stage of development at each time point.3

Type of observation Timing (h post insemination) Expected stage of development
Fertilization check 17 ± 1 Pronuclear (PN) stage
Syngamy check 23 ± 1 Expect 50% to be in syngamy (up to 20% may be at the 2-cell stage)
Early cleavage check 26 ± 1 post ICSI

28 ± 1 post IVF
2-cell stage
Day 2 embryo assessment 44 ± 1 4-cell stage
Day 3 embryo assessment 68 ± 1 8-cell stage
Day 4 embryo assessment 92 ± 2 Morula
Day 5 embryo assessment 116 ± 2 Blastocyst

8.2 Embryo Culture and Timing of Transfer

The study of mammalian preimplantation embryos in vitro became possible as early as the mid-1950s. The first culture medium specifically devised for mammalian (mouse) embryos was described by Whitten in 1956.5 Subsequently, the pioneering work of McLaren and Biggers in 1958 reported that 8-cell mouse embryos cultured until the blastocyst stage would develop into normal young after transfer to the uterus of surrogate mothers.6 Despite those early advances in mouse embryo culture, extended culture of human embryos remained challenging for several years. Initial attempts of culturing human embryos to the blastocyst stage in simplified media resulted in acceptable blastocyst formation rates, but in disappointingly low implantation and pregnancy rates. For this reason, transfer of embryos on day 2 or day 3 remained standard practice in human IVF for almost two decades.8

The first reliable approach for successful extended culture of human embryos depended on the use of sequential culture media, which were designed to meet the changing metabolic and nutritional requirements of the developing embryo. However, there is now renewed interest in the use of a single medium to support development of the embryo during the entire preimplantation period, based on a single formulation. Especially with the implementation of closed culture with time-lapse imaging, single culture media is again gaining ground. Both single and sequential media have been shown to be equally efficient in supporting embryo development to the blastocyst stage, and both approaches seem to be associated with similar reproductive outcomes.10 (See discussion in Chapter 5).

Certainly, blastocyst culture is more technically demanding compared to Day 2/3 culture, and there are several laboratory aspects that should be considered apart from the culture medium. Extended culture up to the blastocyst stage requires greater incubator capacity, which will depend on the number of cycles each clinic accommodates. Moreover, moving embryos to fresh medium on day 3, if sequential media are used, can increase workload in busy laboratories. In addition, air filtration, pH monitoring of culture media, and use of low oxygen are of crucial importance for a successful blastocyst transfer program.

The ability to grow human embryos until the blastocyst stage using new generation media has provided clinics with more flexibility, as now embryo transfers can be performed on all days of preimplantation development. Embryos are usually transferred either at the cleavage stage (on Day 2 or 3) or at the blastocyst stage (on Day 5). Current evidence suggests that blastocyst transfer of Day 6 is associated with lower clinical pregnancy and live birth rates than Day 5, especially for fresh transfers.11 Therefore, it is suggested that slow developing embryos that form a blastocyst on Day 6 are cryopreserved, and transferred in a subsequent frozen replacement cycle on Day 5.12 At present, morula transfer on Day 4 is less common (see discussion in 8.3 The Morula Stage.)

Blastocyst transfer is becoming more popular mainly because it offers several advantages, discussed below, and is theoretically associated with an increased probability of implantation compared to cleavage stage transfer. However, there is still a debate regarding the comparative efficacy of cleavage and blastocyst culture per oocyte pick-up, and in addition some concerns have been expressed regarding the effect of extended culture on perinatal outcomes.

8.2.1 Cleavage-Stage and Blastocyst Transfer Embryo Selection

The cohort of oocytes retrieved from a patient following controlled ovarian stimulation is quite heterogeneous in terms of maturity and developmental competence. Consequently, the resulting embryos have a marked variability in their developmental and implantation potential. Therefore, there is a constant challenge for embryologists to accurately distinguish good quality embryos and select those for transfer. In this respect, embryo morphology assessment has historically been the first line tool for embryo selection. However, morphological assessment is subjective and is characterized by high intra- and inter-operator variability.13 Moreover, embryo morphology on Days 2–3 has been shown to have limited ability to predict blastocyst formation on Day 5 (Figure 8.2).14

Figure 8.2 Pictures a patient’s embryos as they develop from Day 2 (a), to Day 3 (b) to Day 5 (c). Embryo selection on Days 2 and 3 is hindered by the fact that all embryos look equally suitable for transfer. On Day 5, however, the formation of blastocysts allows a more accurate identification of the best embryos.

(Photographs taken by the author).

Blastocyst formation rates usually range from 40% to 60%, meaning that not all embryos have the potential to form a blastocyst.10 Therefore, extending culture to Day 5 is considered to represent a mechanism for self-selection of embryos with higher developmental competence. The blastocyst stage reflects embryos that have successfully made the transition from maternal dependence to embryonic genome activation, while there is also evidence that blastocysts are associated with a reduced incidence of aneuploidies compared to cleavage-stage embryos.15 Single Embryo Transfer and Reduction of Multiple Pregnancies

Faced with our current inability to accurately identify the single best embryo for transfer from a patient’s cohort, in some cases two or more embryos are transferred. As a result, the risk of multiple pregnancies is increased. Multiple pregnancy is the most serious complication of IVF as it carries a higher chance of miscarriage, perinatal mortality, cerebral palsy, hypertension, preeclampsia, gestational diabetes and the need for a caesarean birth.

Moreover, in the UK, it is reported that the average cost to the NHS is £13,959 for a twin birth, compared to £4,892 for a singleton birth. The percentage of multiple pregnancies from IVF in the UK has seen a progressive decline because of changes in policy and the implementation of elective single embryo transfer (eSET). This decrease clearly needs to be sustained. A recent report issued from the Human Fertilization and Embryology Authority (HFEA) shows that for every 10% reduction in the current rate of multiple births, £15 million could be saved by the NHS.

The ultimate goal of efficient and accurate embryo selection is therefore to maximize the probability of selecting the embryo with highest potential first. To this end, improved embryo selection, facilitated by blastocyst culture, may promote the use of eSET, by making feasible the transfer of a single embryo without reducing the patients’ chances of a successful pregnancy.

Current evidence suggests that in good-prognosis patients, transfer of a single blastocyst significantly decreases the incidence of multiple pregnancy, while maintaining pregnancy rates comparable to those following double blastocyst transfer.16 In addition, the decline in multiple birth rates has been shown to have a positive impact on the health of the offspring. Follow-up studies show that perinatal risks for ART children has decreased and overall health has improved.17 Embryo–Uterine Synchronization

The efficiency and flexibility of human IVF has benefited greatly from the ability to transfer embryos at all preimplantation stages (cleavage-stage or blastocyst) to the uterus. It should be noted that, in vivo, cleavage stage embryos reside in the fallopian tube and not in the uterus. Interestingly, in domestic and other mammalian species, transfer of cleavage stage embryos into the uterus results in impaired development and low pregnancy rates, thus necessitating transfer at the morula or blastocyst stage.

In humans, however, transfer of cleavage stage embryos to the uterus does not appear to compromise their development or their ability to implant. Nevertheless, there remains a theoretical advantage that transfer of a blastocyst may allow for a more physiological synchronization of the embryo stage and the uterine environment, possibly leading to improved implantation rates.8 Embryo Biopsy for Preimplantation Genetic Testing

Currently, all preimplantation genetic testing (PGT) programs involve performing an embryo biopsy. Biopsy can be performed by removal of one or two polar bodies, respectively, from the unfertilized oocyte or the zygote, blastomere removal from Day 3 embryos, or removal of five to ten trophectoderm cells at the blastocyst stage during Days 5–7 post-insemination.18

Cleavage stage biopsy involves the removal of a single blastomere of a Day 3 embryo that has at least six blastomeres up to precompaction stages. At these stages, blastomeres have the potential to contribute to the embryo proper since they have not yet committed to either the inner cell mass or trophectoderm. It is possible to detect meiotic errors, but mitotic errors leading to chromosomal mosaicism cannot be identified, and the amount of DNA obtained from a single cell is limited.

Following biopsy, embryos are either further cultured to the blastocyst stage while waiting for the genetic result or are cryopreserved. This latter approach is characterized by increased laboratory workload, as a high number of Day 3 embryos are biopsied, whose potential to develop to the blastocyst stage is still unknown. A negative impact of cleavage stage biopsy on embryo developmental competence has been suggested,19 especially if two cells are removed.20

Blastocyst biopsy involves the removal of 5 to 10 trophectoderm cells. These cells are destined to give rise to the placenta and the extraembryonic membranes and not the embryo proper, thus theoretically inflicting lower impact on the fetus, which originates from the inner cell mass.

While cleavage-stage biopsy was originally the most widely practiced form of embryo biopsy,18 its clinical use has now been reduced. Modern PGT strategies involve biopsy at the blastocyst stage followed by comprehensive chromosome screening and possibly combined with vitrification.19 Blastocyst biopsy is the most widely adopted technique, as it provides higher reliability by the analysis of a higher number of cells, ability to detect chromosome mosaicisms, is associated with lower laboratory workload by biopsying fewer embryos, and is less sensitive to possible embryo damage compared to cleavage-stage biopsy. Embryo Cryopreservation

Embryo cryopreservation has become a vital key component of IVF programs, allowing several embryo transfers from the same oocyte retrieval cycle, leading to an increase in cumulative pregnancy rates. Extended culture to the blastocyst stage and blastocyst cryopreservation has a central role in modern ART strategies. Although slow-freezing seemed to compromise blastocyst cryosurvival, as indicated by lower cumulative pregnancy rates,22 blastocyst vitrification offers excellent survival rates.23 Interestingly, there is only one available randomized control trial (RCT) comparing cleavage-stage and blastocyst cryopreservation using vitrification.24 This study reported significantly higher cumulative pregnancy rates in the blastocyst stage group (Day 3 freezing: 43.3% vs. Day 5 freezing: 56.8%).

The success rates of blastocyst vitrification have significantly increased the efficiency of IVF and have transformed modern clinical practice.25 Furthermore, successful cryopreservation is essential for an effective eSET program, segmentation of treatment to prevent ovarian hyperstimulation syndrome by employing a freeze-all policy,26 and implementation of modern PGT strategies.21

2.1.6 Monitoring of Ovarian Hyperstimulation Syndrome

Ovarian hyperstimulation syndrome (OHSS) is a serious and potentially life-threatening iatrogenic complication of ovarian stimulation. OHSS is triggered by human chorionic gonadotrophin (hCG), which induces excessive ovarian secretion of angiogenic factors, resulting in increased vascular permeability, fluid shift to the third space, and ascites accumulation in the third space and peritoneum. Early-onset OHSS occurs within 5 days after oocyte retrieval and is classified in mild, moderate, and severe forms.27

Generally, in high-risk for OHSS patients, culture to the blastocyst stage is recommended and allows better clinical evaluation of the patient, with the possibility of embryo transfer cancellation, in case severe OHSS develops by Day 5. Usually, patients at high risk for OHSS are women with a good ovarian reserve, who yield high numbers of oocytes and embryos and are therefore suitable for blastocyst culture. Blastocyst transfer may be performed if severe OHSS is not diagnosed on Day 5. However, these patients should be informed about the probability of late pregnancy-induced OHSS and ideally be offered a freeze-all option.28

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Apr 26, 2021 | Posted by in GYNECOLOGY | Comments Off on Chapter 8 – Timing of Embryo Culture
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